1. Field of the Invention
This invention relates to affinity binding assays, such as immunoassays and nucleic acid hybridization assays, in which enzymes are used as labels and the presence or level of a substance is inferred from the enzymatic activity observed.
2. Description of the Background Art
Affinity Binding Assays. Measurement or detection of minute amounts of analytes, especially but not exclusively substances of biological origin, typically relies on processes of molecular recognition to achieve specificity. By molecular recognition is meant a highly specific interaction between two molecules, by virtue of which they bind to each other with high affinity The complex so formed is typically denoted as an "affinity complex"; and one of its members is often designated as a "ligand" of the other. This phenomenon forms the basis of the well known technique of affinity chromatography. Prominent illustrative examples of molecular recognition are antibody-antigen complex formation and hydridization of complementary nucleic acids (cf. Journal of Molecular Recognition for further examples).
Analytical protocols are typically classified as "homogeneous" or "heterogeneous". Heterogeneous assays are those in which at least one component of the affinity complex is necessarily immobilized on a surface during all or part of the analysis, i.e. such immobilization is methodologically functional. Conversely, homogeneous assays do not require immobilization of either member of the affinity complex, i.e. the analysis occurs substantially in solution.
For a homogeneous assay to be successful, there must be a difference between the signal produced by the labeled reagent in the free and in the bound state. Amos, U.S. Pat. No. 4,442,218 (1984) permits a binding reaction to take place within the pores of an insoluble porous monolith. The signal produced by the labeled species, once bound within the pore, is attenuated relative to the signal emitted by the free label species. Zuk, U.S. Pat. No. 4,256,834 employs three reagents: an insolubilized ligand, a labeled (e.g. fluorescein) anti-ligand, and an insolubilized anti-label (e.g., anti-fluorescein antibody bound to charcoal). When the first two reagents form an affinity complex, the third, the signal repressor, is obstructed from interacting with the label. Free labeled anti-ligand, however, is complexed by the anti-label and its signal production is inhibited. Zuk, U.S. Pat. No. 4,281,061, discloses an assay which features a labeled ligand, an antiligand, an anti(antiligand), and a polyvalent macromolecule which can modulate the signal produced by the labeled ligand. Ligand, antiligand, and anti(antiligand) bind to form a matrix which either encloses the macromolecule (resulting in enhanced interaction) or excludes it (resulting in diminished interaction). The macromolecule is typically an antibody. Carrico, U.S. Pat. No. 4,743,535 (1988) teaches use of an antibody which binds to double-stranded but not single stranded DNA in a nucleic acid hybridization assay. The antibody sterically interferes with signal generation by the affinity complex. Halfman, U.S. Pat. No. 4,640,898 forms micelles which can distinguish unbound labeled conjugate and the conjugate as part of an affinity complex. See also Rubenstein U.S. Pat. No. 3,817,837; Ullman, U.S. Pat. No. 3,996,345; Rubenstein, U.S. Pat. No. 3,935,074.
Homogeneous assays offer several advantages relative to heterogeneous assays, including the following:
i) no immobilization step is required; PA1 ii) time-consuming and labour-intensive washing steps are unnecessary; PA1 iii) capable of performance in a simple, reusable container such as a test tube, by addition of reagents without liquid withdrawal; PA1 iv) more rapid analysis due to improved mass-transfer kinetics.
In spite of these advantages very few operational homogeneous assay systems have been developed to-date. An important advantage of the present invention is that it functions in either homogeneous or heterogeneous assay formats. Moreover homogeneous assays employing the invention complement existing technology which can only measure analytes that are small molecules (Chan, 1987).
Another functional distinction is between competitive and non-competitve assays. A competitive assay relies on at least one process of competition between labelled and unlabelled species (e.g. analyte, antibody, DNA probe, etc.) for binding to a complementary molecule through a process of molecular recognition. Conversely a non-competitive assay requires only binding of a labelled probe to the analyte by a molecular recognition process. Because of thermodynamic constraints competitive assays are typically about one thousand-fold less sensitive than non-competitive assays: "Thus, the detection limit of antigens by competitive immunoassay is at femtomole [10.sup.-15 mol] or higher levels in most cases...The detection limit of antigens by non-competitive two-site enzyme immunoassay with appropriate techniques is at attomole [10.sup.-18 mol] levels." (Ishikawa et al , 1991).
In spite of these recognized advantages of non-competitive, homogeneous assays, it appears that no such systems have been developed to the point of practical utility. Thus, "Although competitive assays were used in early EIA [enzyme immunoassay] there has subsequently been a progressive increase in the use of non-competitive or immunoenzymometric assays...These assays all use solid-phase reagent, either an antibody or antigen." (Gould and Marks, 1988). Similarly all of the homogeneous immunoassay methods described by Ngo (1985) are of the competitive type. A significant advantage of the present invention is its general applicability in a non-competitive, homogeneous assay format, thereby deriving most or all of the benefits described above.
Assay Sensitivity. Heretofore detection methods relying on molecular recognition processes have suffered from compromises between detection limit and analysis time. Thus, "...although the theoretical detectability of catalyst [sic] could be as low as a single molecule, practical considerations of time and the inevitability of backgrounds have limited the sensitivity of enzyme-based assays." (Bates, 1987). The detection limit of heterogeneous assays has always been constrained by a non-specific, "background" (zero analyte) signal. Although to some extent reducible, such background has not heretofore been susceptible to substantial elimination: "In two-site enzyme immunoassay for antigen, enzyme-labelled antibody non-specifically (non-immunologically and physically) adsorbs to antibody-coated solid phase to various extents. This is one of the major obstacles to improvement in sensitivity and various attempts have been made to reduce the non-specific binding." (Ishikawa et al., 1991). None of these previous attempts have met with unqualified success.
The various sources of background signal were described as follows by Pruslin et al. (1991): "The OD (absorbance) measurements obtained in an assay include, in addition to the values for the specific antibody/antigen reaction, summary values designated `background`. That designation includes all contributions to the OD that are not attributable to the specific antibody/antigen reaction under test. Those non-specific contributions are demonstrable by including a set of test wells with serum (antibody) but with antigen omitted, and a set of wells with the series of antigen concentrations, but with serum omitted.
In the first instance, the revealed `background` may be due to reactivity of antibodies, other than the test antibody, with antigenic moieties in the reagents or to non-reactive serum components that are detected by the enzyme-labeled probe, second antibody. For the second set of `control` wells, those with serum omitted, the revealed `background` may be due to impurities in the antigen solution with which the probe antibody is reactive or to adventitious deposits of the enzyme-labeled probe on the well surface."
Considerable efforts have been expended to minimize the background signal in immunoassays. Present methods employed to this end include rigorous washing with surfactants (Mohammad and Esen, 1989), application of protein solutions to block non-specific adsorption sites (Pruslin et al., 1991), and careful selection of solid-phase and solution-phase antibodies (Aoyagi et al., 1991). In spite of such measures background interference remains, and complex and not entirely unambiguous calculations to compensate for same are necessary (Pruslin et al., 1991).
In order to reduce the fundamental limitation of non-specific background signal interference several reversible capture or transfer methods have been developed. These methods are all characterized by at least one transfer of analytical markers, especially the analyte itself, between two surfaces, generally for the purpose of background reduction. Lejeune et al. (1990) detected about 1.times.10.sup.-19 mol of human growth hormone in four hours using two immunoaffinity resins. Ishikawa et al. (1991) describe methods developed to "transfer the complex of analytes and labelled reactants from solid phase to solid phase without dissociation." These repetitive and labour intensive procedures permit the measurement of about 1.times.10.sup.-21 mol (about 600 molecules) of antigen in 15 to 40 hours. Nevertheless, "One of the greatest obstacles limiting the sensitivity of non-competitive solid phase enzyme immunoassays is the non-specific binding of enzyme-labelled reactants to solid phase." (ibid.) Similar reversible capture methods using the avidin-biotin interaction were recently advocated for background reduction prior to polymerase chain reaction (PCR) amplification of DNA (Yolken et al., 1991).
Direct unamplified measurement of small amounts of specific nucleic acids or oligonucleotides suffers from the same compromise between detection limit and analysis time as found with present immunochemical methods. Molecular recognition of complementary DNA strands (hybridization) was used by Syvanen et al. (1986) to detect as few as 4.times.10.sup.5 molecules of nucleic acid in 3 hours. Similarly, 6.times.10.sup.4 molecules of viral DNA were detected in 4 hours by use of an oligonucleotide (i.e. nucleotide polymer) probe coupled to enzyme (Urdea et al., 1987). Such methods suffer from interference due to high background signal when applied to crude samples (Hames and Higgins, 1985). Thus Syvanen et al. (1986) could detect no fewer than 5.times.10.sup.5 cells; and a detection limit of 2.times.10.sup.7 bacterial cells in 4.5 to 5 hours was reported by Kennedy et al. (1989). The recognized limitations of direct methods of nucleic acid detection led to the development of various DNA amplification schemes.
Enzymatic Amplification of Signal. Another method used to increase sensitivity is enzymatic amplification of the initial signal. Direct (unamplified) immunochemical analysis faces fundamental limitations due to equilibrium, kinetic and mass transport constraints. Thus, "while improvement in sensitivity and detection limit might always be made in principle by choosing an antibody-antigen pair with higher affinity, this improvement is made at the expense of an increased response time." (Eddowes, 1987/88). Although about 3.times.10.sup.-15 mol of substance per ml are detectable in less than one hour (Liabakk et al., 1990), detection of 7.times.10.sup.-17 mol of substance requires 18 hours (Aoyagi et al., 1991). The practical limit for detection of Salmonella by direct immunochemical assay was recently reported as 10.sup.3 to 10.sup.4 cells/ml in 2 to 3 hours (Luk and Lindberg, 1991).
Enzymatically amplified immunoassays employing alkaline phosphatase combined with cofactor cycling have been reported to be capable of detecting 10.sup.5 molecules during long incubation times (Bates, 1989). More recently less than 10.sup.-19 mol (about 6.times.10.sup.4 molecules) of substance have been detected in 2 hours, but ten thousand times this amount had to be present for detection within an hour (Durkee et al., 1990).
Several immunochemical detection or measurement methods employing enzymatic amplification are disclosed in the patent literature. Eur. Pat. No. 123,265 (Hall and Hargreaves, 1983) teaches that signal amplification can be achieved by covalent coupling of an analyte molecule to a zymogen such as trypsinogen. In U.S. Pat. No. 4,463,090 (Harris, 1984) signal amplification is obtained by a 3- or 4-stage enzyme cascade initiated by an enzyme or zymogen coupled to an immuno-reactive substance. This patent stipulates that it is necessary to use a different enzyme/zymogen pair at each amplification stage, thus limiting the amplification effect to a 3 or 4-stage geometric increase. In U.S. Pat. No. 4,598,042 (Self, 1981) it is disclosed that signal amplification can be achieved when the product of a primary enzyme, especially alkaline phosphatase, participates in a secondary enzyme cycle. U.S. Pat. No. 4,745,054 (Rabin et al., 1988) teaches that two inactive fragments can be combined to form an active enzyme which then activates a second enzyme leading to a detectable result. None of these enzyme cascade methods exploit the most effective type of amplification, namely an exponential increase with time.
Although heretofore not applied to amplification of enzymatic activity coupled to a substance capable of molecular recognition of an analyte, exponential amplification is well known in the biochemical literature. For example, trypsinogen (zymogen) formed in the pancreas of mammals is converted to trypsin by traces of trypsin or enteropeptidase in the intestine (Moss et al., 1987). This so-called "autocatalytic" process produces an exponential increase in the amount of enzyme (Pechere and Neurath, 1957). This principle has been used to measure low concentrations of trypsin after amplification by activation of trypsinogen (Mayer et al., 1974; Aisina et al., 1975; Kazanskaya et al., 1983).
Several strategies for ultra-sensitive detection of nucleic acids based on their exponential amplification have recently been reviewed (Kwoh and Kwoh, 1990). The polymerase chain reaction (PCR) amplification method has rapidly achieved wide acceptance in research applications in spite of its numerous limitations and disadvantages (Lizardi and Kramer, 1991). While PCR methodology has in principle a detection limit of a single oligonucleotide, it suffers from the heretofore universal problem of background interference, as well as from contamination by amplifiable DNA (Kwok and Higuchi, 1989). Furthermore the procedure requires several hours to achieve significant amplification using dedicated and sophisticated equipment. Although detection of 1 to 2 cells has been accomplished in a 5 to 7 hour period, complex manipulations are required for analysis of actual crude samples (Bej et al., 1990). Finally, since the amplification yield in each cycle varies unpredictably, PCR is not well suited for quantitative measurement of nucleic acid concentration (Lizardi and Kramer, 1991). Since PCR analyses for ribonucleic acid (RNA) simply add an initial RNA to DNA transcription step they suffer from all of the above disadvantages (Rose, 1990).
Q-beta amplification, another nucleic acid analysis method under development, has a detection limit of about 2,000 molecules of DNA, but also suffers from several disadvantages (Lizardi and Kramer, 1991). Specifically this technique requires repetitive, multi-step, mass-transfer limited operations to reduce the background signal level prior to amplification. Although amplification with Q-beta replicase is rapid, these multiple hybridization steps appear to impose a minimum analysis time of several hours. Furthermore, contamination and background interference are once again limitations: "A problem that remains to be solved in order to reach the theoretical detection limit of one molecule is the removal of the few hundred probe molecules that are carried along non-specifically with the probe-target complexes during washing, despite reversible target capture. These non-specifically bound probe molecules are also amplified, giving rise to a background signal". (ibid). Finally, as for the PCR technique, dedicated and sophisticated equipment is required.
Signal modulation or amplification is further described in Mize, U.S. Pat. No. 4,835,099, Mapes, U.S. Pat. No. 4,904,583, Schulte, U.S. Pat. No. 4,962,024, Boguslaski, U.S. Pat. No. 4,791,055, Faring, U.S. Pat. No. 4,785,080, and Perlman, U.S. Pat. No. 4,810,631.
Use of Enzyme Inhibitors in Affinity Binding Assays. Enzyme inhibitors have been used as binding reagents (see March, U.S. Pat. No. 4,430,264; Gattaz, U.S. Pat. No. 5,006,462; Newman, U.S. Pat. No. 5,057,430) and as labels (Kasahara, U.S. Pat. No. 4,582,792 and U.S. Pat. No. 4,649,105; Taguchi, U.S. Pat. No. 5,035,995; Bloch, U.S. Pat. No. 4,789,630; Hakomori, U.S. Pat. No. 4,904,596; Matsuura, U.S. Pat. No. 4,894,326; Skov, U.S. Pat. No. 4,869,875; Navinski, U.S. Pat. No. 4,843,010; Maggio, U.S. Pat. No. 4,828,981; Stavrianopoulos, U.S. Pat. No. 4,772,548). See also Bauma, U.S. Pat. No. 5,006,473. Several patents teach use of an enzyme inhibitor to selectively inhibit the activity of an enzyme label. See Ito, U.S. Pat. No. 4,966,856 and 4,868,106; Leeder, U.S. Pat. No. 4,837,395; Gedden, U.S. Pat. No. 4,810,635; Ullman, U.S. Pat. No. 4,913,983; Yoshida, U.S. Pat. No. 4,341,866.
Yoshida, U.S. Pat. No. 4,208,479 teaches an immunoassay which involves bringing together an analyte and a labeled receptor so as to form a complex which sterically inhibits the approach of a macromolecular modifier, which otherwise would physically or chemically interact with the label to reduce its signal production. Where the label was an enzyme the disclosed modifiers were principally anti-(enzyme) antibodies and certain small, organic, irreversible inhibitors. For example, for serine proteases, the preferred inhibitor was physostigmine. Similar inhibitor systems were set forth for use with fluorescent labels. The teachings of Yoshida, U.S. Pat. No. 4,233,401 and Yoshida, U.S. Pat. No. 4,341,866 are similar.
Louderback, U.S. Pat. No. 4,668,0630, teaches use of a reversible inhibitor to protect an enzyme (lactate hydrogenase).
Alpha2-Macroglobulin. Assays for alpha2-macroglobulin have been described, see Siddiqui, U.S. Pat. No. 4,607,010, but A2M has not previously seen service as a signal modulator in a binding assay. A2M is also mentioned in Silvestrini, U.S. Pat. No. 5,047,509; Lorier, U.S. Pat. No. 5,013,568; Harpel, U.S. Pat. No. 4,629,694; Teodorescu, U.S. Pat. No. 4,499,186; and Schrenk, U.S. Pat. No. 4,414,332 and U.S. Pat. No. 4,393,139.